World Journal of Agricultural Sciences 3 (3): 339-343, 2007
ISSN 1817-3047
© IDOSI Publications, 2007
Corresponding Author: Dr. E.M. Ilondu, Department of Botany and Microbiology, Delta State University, Abraka Delta State,
Nigeria
339
Inhibition of Three Fungal Isolates from Sorrel Drink (Zobo) Using Hurdle Technique
E.M. Ilondu and A.C. Iloh 1 2
Department of Botany and Microbiology, Delta State University, Abraka Delta State, P.M.B. 1, Nigeria 1
Bioresources Development and Conservation Programme (BDCP), Wuse II, Abuja, Nigeria 2
Abstract: Studies were carried out on the inhibition of three fungi isolated from sorrel drink (Zobo) using water
extracts of Zingiber officinale (Ginger), Piper guineense (West African Black Pepper) as well as heat as hurdles.
The fungi isolated and identified were Aspergillus flavus, A, niger and unidentified Yeast. The spice extracts
at concentrations 1-6 ml reduced fungal growth. As the spice concentration was increased, there was a decrease
in fungal biomass in the drink when compared with the control. A combination of both spice extracts at different
ratios also reduced fungal growth. When the fungi were challenged with 100°C heat for different periods of
times (mins), growth reduction also occurred. The further combination of heat treatment with the combined
spice treatment inhibted growth tremendously.
Key words: Aspergillus flavus % A. niger % Growth inhibition % Hurdle technology % Piper guineense (West
African black pepper) % Sorrel drink (Zobo) % Yeast % Zingiber officinale (Ginger)
INTRODUCTION and nutrition. In this regard, many investigators
Roselle (Hibiscus sabdariffa L) is a leafy vegetable of or protect food from contamination with harmful
the Malvaceae family [1]. It is locally called Zoborodo microorganisms.
(Hausa) and Sorrel in English. Hibiscus sabdariffa is The term “Hurdle Technology” preservation was
native to India but was introduced to Central America and derived from the use of combination of preservation
West Indies where it became popular in Jamaica at the techniques (hurdles) that any micro-organism present
beginning of the Eighteenth century [2]. Hibiscus would not be able to overcome [5]. This method has
sabdariffa is adapted to a wide range of soil conditions become popular because the hurdles are used at low
and is often grown on relatively infertile soils but concentrations which do not have effect on taste while
economic yields are obtained on soils which are well maintaining the stability and safety of the food. Hurdles
supplied with organic matter and essential nutrients [3]. In for preservation according to Leistner and Hachelman [6]
Nigeria, the green type is generally common in the derived include temperature (low and high), pH. (low and high),
and Southern Guinea Savanna while the brown type is water activity, redox potential etc.
more prevalent in Northern Guinea and Sudan savannas. In food preserved with hurdle technology, the
The tender leaves and stalks are eaten as salad and as a possibility exists that different hurdles may not only have
pot herb and are used for seasoning with curry. The seeds an additive effect on the stability but act synergistically.
contain oil and are eaten in Africa [4]. The seeds are also A synergistic effect could result, if the hurdles in food hit
boiled, fermented and dried for use as condiments for different targets e.g. cell membrane, DNA etc. Thus, it is
local soup preparations (Yakuwa or Batso in Hausa) [4]. more effective to use different preservatives in small
Again the red succulent calyx are boiled with sugar to amounts than only one in large amounts because the
produce a local drink called Zobo drink. This drink has former might hit different targets within the microbial cell
become a household name in Nigeria, it is also very synergistically [7].
nutritious. However, Zobo, like any other drink or food is Hurdle Technology approach has been applied by
susceptible to deterioration by food borne microbes, this David and Shibamoto [8] in an attempt to preserve peach,
reduces the quality of the drink in terms of colour, taste pineapple, mango slices and banana puree using slight
recommended the use of food preservatives to disinfect
Number of fungus colony encountered
100
Total number of all fungal colonies
´
World J. Agric. Sci., 3 (3): 339-343, 2007
340
reduction in water activity, low pH and addition of anti- Ogbogonogo market in Asaba, Delta State and prepared
microbials. Fresh okra was successfully stored using three by the method of Akpomedaye and Ejechi [11]. Samples
hurdles comprising two spices and mild heat [9]. were ground into powder and extracted with hot sterile
Ogiehor et al. [10] preserved the African Oil bean water (80°C at 5g/100 ml for 72 hours). Extracts were
using a combination of temperature and sodium chloride. filtered using sterile whatman No. 1 filter papers. The
Akpomedaye and Ejechi [11], Ejechi et al. [12] and Ilondu spice extracts were concentrated through exposure to
et al. [13] have used various combinations of spices and 100°C for 2 mins in a water bath.
mild heat to preserve fruit juices and Jam produced from
velvet tamarind. Effects of the spice extracts on fungal growth in Zobo
Therefore an attempt to use this technology of two drink: To each 20ml of the Zobo drink, 1-6ml of either
spices Ginger and West African Black pepper as well as spice extract was added plus water to make up 6ml volume
heat to inhibit three fungal isolates from Zobo drink was as control. This was inoculated with 1ml of 10 spore
the target of the present work. It is expected that this suspension obtained from serial dilution from one week
would develop a method that would enhance the shelf life old cultures of each test fungi. The samples and the
of Zobo drink. control (with 6ml water) were incubated for 2 weeks at
MATERIALS AND METHODS harvested by filtration through sterile Whatman No.1 filter
Experimental studies were carried out at the Botany Biomass (dry weight) was then determined using an
and Microbiology Laboratory of the Delta State electronic balance.
University Abraka, Nigeria.
Collection of material and preparation of Zobo drink: the drink were prepared and autoclaved. The same
Dried calyces of H. sabdariffa, (red variety) were procured procedures were repeated using combinations of the two
from Ogbogobogo market in Asaba, Delta state. The Zobo spice extracts in the following v/v proportions ginger and
drink was prepared according to the local procedures by black pepper: 1:1, 2:1, 1:2 and 2:2. Samples free of spice
adding 350g of dry calyces into 1000ml of boiling tap extracts (replaced with sterile water in the usual way) were
water and allowed to stand for 1hr. The calyces were used as control and all treatments were replicated three
removed by filtration using a sterile sieve cloth; and 20g times.
of granulated sugarwas added. This freshly prepared
drink was then dispensed into sterile 200ml bottles and Effect of mild heat: Autoclaved Zobo drink was
left at room temperature (30±2°C) for 14 days. inoculated with 1ml of 10 spore suspension of each of the
Isolation and identification of associated fungal [12], bottles were placed on a water bath at 100°C for
organisms: After 14 days, I ml sample was plated on different periods of time: 1, 2, 3, 4 and 5 min. The
potato dextrose agar (PDA) amended with experiments were then kept at room temperature (30±2°C)
chloramphenicol (0.5 mg mlG ). The Petri plates were then and mycelial growth was assessed after 2 weeks. 1
incubated for 72 hours at room temperature (30±2°C). The
resulting mycelial growth was transferred to fresh PDA Effect of combination of spice extracts and heat:
plates for further growth and identification by microscopic Autoclaved zobo drink was inoculated with I ml of 10
examination with the aid of Barnett and Hunter [14]. spore suspension of each test fungus. Bottles of samples
Percentage frequency of occurrence of isolated fungi was were placed on a water bath at 100°C for 1-5mins before
determined using the formula: the introduction of the combination of 2:2 of the spice
(30±2°C) and growth assessed after 2 weeks [12].
Collection and extraction of spices: Dried spice samples
of ginger (Zingiber officinale) and West African black The identified fungi reported in this paper as
pepper (Piper guineense) were procured from associated with Zobo drink were Aspergillus flavus, A.
5
room temperature (30±2°C). The resulting mycelia were
paper and dried to a constant weight at 70°C for 5mins.
Effects of spice extract combinations: Fresh samples of
5
test fungi. Following the method described by Ejechi et al.
5
extracts. The experiments were kept at room temperature
RESULTS AND DISCUSSION
World J. Agric. Sci., 3 (3): 339-343, 2007
341
Table 1: Percentage frequency of occurrence of fungal isolates associated Table 4: Fungal growth in Zobo drink in response to heat treatment at
with Zobo drink 100°C for different periods of time
Fungus Total No. of isolation Frequency (%) Dry wt. of fungal biomass (mg)
Aspergillus flavus 24 32
A. niger 22 28
Yeast 30 40 0.0 (Control) 27±1.0 56±1.5 22±1.0
Table 2: Effect of aqueous extracts of ginger and West African black pepper
on fungal growth in Zobo drink
Dry wt. of fungal biomass (mg)
-------------------------------------------------------
Extract concentration (ml) A flavus A. niger Yeast
Ginger extract
0.0 (Control) 27±1.0 56±1.5 22±1.0
1.0 28±1.0 54±1.0 19+0.6 a a a
2.0 24±1.5 50±1.0 17±1.0 b b b
3.0 22±1.5 35±1.0 10±1.0 b c c
4.0 19±1.0 32±1.5 10±1.6 c c c
5.0 16±0.6 28±1.5 8±1.0 c c c
6.0 13±1.0 18±1.0 6±1.0 c c c
West African black pepper extract
0.0 27±1.0 56±1.5 22±1.0
1.0 27±0.5 54±1.5 19±0.6 a a a
2.0 24±1.0 34±2.0 17±1.0 b c b
3.0 22±0.2 28±1.5 10±1.0 b c c
4.0 20±1.0 25±2.1 10±1.6 b c c
5.0 18±1.5 21±1.6 8±1.0 c c c
6.0 14±1.5 1.5±2.0 6±1.0 c c c
Significant level of difference from the control, a: p>0.05, b: p<0.05, c:
p<0.01
Table 3: Effect of different combinations of standard aqueous extracts
of ginger and West African black pepper on fungal growth
in Zobo drink
Spice extract (ml) Dry wt. of fungal biomass (mg)
------------------------------------------------- --------------------------------------------
Ginger West African black pepper A flavus A. niger Yeast
0.0 0.0 27±1.0 56±1.5 22±1.0
1.0 1.0 25±1.0 48±1.5 22±1.0 a b a
2.0 1.0 22±1.5 40±1.0 16±1.0 b b b
1.0 2.0 21±1.0 42±1.5 12±1.5 b b b
2.0 2.0 19±1.0 30±1.0 8±1.0 c c c
Significant level of difference from the control, a: p>0.05, b: p<0.05, c:
p<0.01
niger and Yeast with the percentage frequency of 32, 28
and 40% respectively (Table 1). These organisms were
easily trapped during the post-harvest processing of the
calyces of H. sabdariffa. Since most fungal spores are
found in the air, the spores must have fallen on the calyx
--------------------------------------------------------
Duration of time (mins) A flavus A. niger Yeast
1.0 22±1.0 30±0.6 15±1.0
2.0 20±1.0 23±1.0 13±.06 a b a
3.0 17±1.0 19±1.0 11±1.0 b b b
4.0 10±0.6 15±1.0 8±1.0 b b b
5.0 5±0.6 10±1.0 4±1.0 c c c
Significant Level of difference from the control, a: p>0.05, b: p<0.05, c:
p<0.01
Table 5: Effect of combination of ginger and black pepper spice extracts (2:2
ratio) and 100°C heating on fungal growth in Zobo drink for
varying periods of time
Dry wt. of fungal biomass (mg)
--------------------------------------------------------
Duration of time (mins) A flavus A. niger Yeast
(Control) 0 27±1.0 56±1.5 22±1.0
1 10±2.0 24±1.0 10±1.0
2 10±1.0 20±1.0 7±1.0 a a a
3 4±0.6 13±1.5 5±0.1 b b b
4 2±1.0 6±1.0 1±1.0 d c d
5 0±0.0 1±1.0 0±0.0 d d d
Significant level of difference from the Control, a: p>0.05, b: p<0.05, c:
p<0.01, d: p<0.00
during drying. Dongo and Ayodele [15] have shown that
Aspergillus occurred highest in the number of colonies
identified from air spora of some localities in Delta State
University, Abraka. The spores liberated in the air can
quickly settle on food, ceilings of room and then
germinate [16].
Data in Table (2) showed the effect of water extracts
of Ginger and West African Black Pepper on fungal
growth in Zobo drink. As the concentration of the spice
extracts was increased, a remarkable decrease in the
fungal biomass was recorded. Combination of the spice
extracts showing the highest reduction of fungal growth
was 2:2 ratio (Table 3). Data in Table (4 and 5) showed the
effects of heat (100°C) of different periods of time (mins)
and in combination with spice extract at 2:2 ratio on the
growth of the test fungus. Results showed a drastic
reduction in biomass of the test organisms when the
exposure time was extended to 5 mins. This is not the first
report showing protection of fruit juices from
biodeterioration using hurdle technology or combined
processes [11-13].
World J. Agric. Sci., 3 (3): 339-343, 2007
342
Nakatani, [17] and Ilondu et al. [13] reported that REFERENCES
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properties of the spices ginger and black pepper. Also
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test organisms. The application of only heat would have
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hurdle was difficult for the organisms to overcome.
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growth at 2:2 spice extract combination and 100°C heat for
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In conclusion, this work has shown that there is
drastic growth inhibition of the test organisms by
combination of spices and heat treatment. These spices
can be obtained locally and are very inexpensive hence
justifying the statement made by Leistner [7] that
preservation in developing countries should be
inexpensive and simple but very reliable. Besides the
antimicrobial activities, spices add flavour which can make
the Zobo drink more consumer friendly.
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